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Analysis of the profile of the tRNA modifications in severalArchaeaallowed us to observe a novel modified uridine in the V-loop of several tRNAs from two species:Pyrococcus furiosusandSulfolobus acidocaldarius. Recently, Ohira and colleagues characterized 2′-phosphouridine (Up) at position 47 in tRNAs of thermophilicSulfurisphaera tokodaii, as well as in several other archaea and thermophilic bacteria. From the presence of the genearkIcorresponding to the RNA kinase responsible for Up47 formation, they also concluded that Up47 should be present in tRNAs of other thermophilicArchaea. Reanalysis of our earlier data confirms that the unidentified residue in tRNAs of bothP. furiosusandS. acidocaldariusis indeed 2′-phosphouridine followed by m5C48. Moreover, we find this modification in several tRNAs of otherArchaeaand of the hyperthermophilic bacteriumAquifex aeolicus.

The 5′UTR part of coronavirus genomes plays key roles in the viral replication cycle and translation of viral mRNAs. The first 75–80 nt, also called the leader sequence, are identical for genomic mRNA and subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5′UTR segment and the nonstructural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5′UTR RNA segment and the NSP1 protein have been done for several coronaviruses, with special attention to the betacoronaviruses. The conclusions are: (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein, indicating a coevolution between the 5′UTR and NSP1 in betacoronaviruses. Experimental mutational data on 5′UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support these conclusions and show that some conserved key residues in the amino-terminal half of the NSP1 protein are essential for evasion to the inhibitory effect of NSP1 on translation.

The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report double-blind assessments of RNA structure predictions in CASP15, the first CASP exercise in which RNA modeling was assessed. Forty two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, who did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and X-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global topology of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as non-canonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.

Continuous and imaginative technological developments are leading to a massive accumulation of various types of data in all areas of biological research. As a result, the central importance of databases is increasing. Databases related to biology must not only be structured using controlled vocabularies, but also be fully integrated into the whole biological domain. To achieve this goal, they must be systematically grounded in biological evolution and exploit the available tools of evolutionary systematics to contribute to our understanding of life processes.

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8–P4 and L18–P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8–P4 and L18–P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9–P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.

tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb database (the Lowe Laboratory, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for alanine compared to other tRNA species, including tRNAs specific for glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair generally observed as a Watson–Crick pair throughout phylogeny. Second, the tertiary pair R15/Y48 occurs as a purine–purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine–purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to a variable extent depending on phylogeny. Among eukaryotes, insects display all variations simultaneously, whereas mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, nor archaeal, tRNAs, probably because of the avoidance of A34-containing anticodons in four-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect, besides aminoacylation, transcription rates (e.g., 54/58) or ribosomal translocation (30/40).

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G+3 in the mRNA. In such pairs the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

The outbreak of the COVID-19 pandemic has led to intensive studies of both the structure and replication mechanism of SARS-CoV-2. In spite of some secondary structure experiments being carried out, the 3D structure of the key function regions of the viral RNA has not yet been well understood. At the beginning of COVID-19 breakout, RNA-Puzzles community attempted to envisage the three-dimensional structure of 5′- and 3′-Un-Translated Regions (UTRs) of the SARS-CoV-2 genome. Here, we report the results of this prediction challenge, presenting the methodologies developed by six participating groups and discussing 100 RNA 3D models (60 models of 5′-UTR and 40 of 3′-UTR) predicted through applying both human experts and automated server approaches. We describe the original protocol for the reference-free comparative analysis of RNA 3D structures designed especially for this challenge. We elaborate on the deduced consensus structure and the reliability of the predicted structural motifs. All the computationally simulated models, as well as the development and the testing of computational tools dedicated to 3D structure analysis, are available for further study.

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

In silico prediction is a well-established approach to derive a general shape of an RNA molecule based on its sequence or secondary structure. This paper reports an analysis of the stereochemical quality of the RNA three-dimensional models predicted using dedicated computer programs. The stereochemistry of 1,052 RNA 3D structures, including 1,030 models predicted by fully automated and human-guided approaches within 22 RNA-Puzzles challenges and reference structures, is analysed. The evaluation is based on standards of RNA stereochemistry that the Protein Data Bank requires from deposited experimental structures. Deviations from standard bond lengths and angles, planarity or chirality are quantified. A reduction in the number of such deviations should help in the improvement of RNA 3D structure modelling approaches.

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNA(ProA), that are predominately found in plant pathogens from Streptomyces species. We then show that tRNA(ProA) has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNA(ProA) can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

Significant improvements have been made in the efficiency and accuracy of RNA 3D structure prediction methods during the succeeding challenges of RNA-Puzzles, a community-wide effort on the assessment of blind prediction of RNA tertiary structures. The RNA-Puzzles contest has shown, among others, that the development and validation of computational methods for RNA fold prediction strongly depend on the benchmark datasets and the structure comparison algorithms. Yet, there has been no systematic benchmark set or decoy structures available for the 3D structure prediction of RNA, hindering the standardization of comparative tests in the modeling of RNA structure. Furthermore, there has not been a unified set of tools that allows deep and complete RNA structure analysis, and at the same time, that is easy to use. Here, we present RNA-Puzzles toolkit, a computational resource including (i) decoy sets generated by different RNA 3D structure prediction methods (raw, for-evaluation and standardized datasets), (ii) 3D structure normalization, analysis, manipulation, visualization tools (RNA_format, RNA_normalizer, rna-tools) and (iii) 3D structure comparison metric tools (RNAQUA, MCQ4Structures). This resource provides a full list of computational tools as well as a standard RNA 3D structure prediction assessment protocol for the community.

Some of the amazing contributions brought to the scientific community by the Protein Data Bank (PDB) are described. The focus is on nucleic acid structures with a bias toward RNA. The evolution and key roles in science of the PDB and other structural databases for nucleic acids illustrate how small initial ideas can become huge and indispensable resources with the unflinching willingness of scientists to cooperate globally. The progress in the understanding of the molecular interactions driving RNA architectures followed the rapid increase in RNA structures in the PDB. That increase was consecutive to improvements in chemical synthesis and purification of RNA molecules, as well as in biophysical methods for structure determination and computer technology. The RNA modeling efforts from the early beginnings are also described together with their links to the state of structural knowledge and technological development. Structures of RNA and of its assemblies are physical objects, which, together with genomic data, allow us to integrate present-day biological functions and the historical evolution in all living species on earth.

In silico prediction is a well-established approach to derive a general shape of an RNA molecule based on its sequence or secondary structure. This paper reports an analysis of the stereochemical quality of the RNA three-dimensional models predicted using dedicated computer programs. The stereochemistry of 1052 RNA 3D structures, including 1030 models predicted by fully automated and human-guided approaches within 22 RNA-Puzzles challenges and reference structures, is analyzed. The evaluation is based on standards of RNA stereochemistry that the Protein Data Bank requires from deposited experimental structures. Deviations from standard bond lengths and angles, planarity, or chirality are quantified. A reduction in the number of such deviations should help in the improvement of RNA 3D structure modeling approaches.

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SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by thecis-acting RNA hairpin SL1 in the 5’UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5’UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine ‘Achille heel’ of the virus.

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2′-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations., Nonsense mutations can be corrected by several molecules that activate readthrough of premature termination codon. Here, the authors report that 2,6-diaminopurine efficiently corrects UGA nonsense mutations with no significant toxicity.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited., The recognition of the mRNA codon by the tRNA anticodon is crucial for protein synthesis. Here the authors introduce non-standard nucleotides in bacterial and eukaryotic mRNA to reveal the minimal hydrogen bond requirement of codon-anticodon interaction for efficient and accurate translation.

Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA–RNA or RNA–protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA–RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid–RNA interactions.

International audience; We have previously shown that 5′ halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5′ halves can form homodimers or heterodimers with tRNAGlu 5′ halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5′ halves inside cells. Considering recent reports have shown that 5′ tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.

The wealth of the combinatorics of nucleotide base pairs enables RNA molecules to assemble into sophisticated interaction networks, which are used to create complex 3D substructures. These interaction networks are essential to shape the 3D architecture of the molecule, and also to provide the key elements to carry molecular functions such as protein or ligand binding. They are made of organised sets of long-range tertiary interactions which connect distinct secondary structure elements in 3D structures. Here, we present a de novo data-driven approach to extract automatically from large data sets of full RNA 3D structures the recurrent interaction networks (RINs). Our methodology enables us for the first time to detect the interaction networks connecting distinct components of the RNA structure, highlighting their diversity and conservation through non-related functional RNAs. We use a graphical model to perform pairwise comparisons of all RNA structures available and to extract RINs and modules. Our analysis yields a complete catalog of RNA 3D structures available in the Protein Data Bank and reveals the intricate hierarchical organization of the RNA interaction networks and modules. We assembled our results in an online database (http://carnaval.lri.fr) which will be regularly updated. Within the site, a tool allows users with a novel RNA structure to detect automatically whether the novel structure contains previously observed RINs.

Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented ‘double K-turn’ module in type A and type M archaeal RPR variants.

The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity., Metal ions play essential roles in myriads of biological processes, from catalytic co-factors to supporting protein and nucleic acid structures. Here the authors use long-wavelength X-ray diffraction to locate hundreds of potassium ions taking part in the formation of rRNA tertiary structure, mediating rRNA–protein interactions and supporting ribosomal protein structures and function.

Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10−4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum., Communications Biology, 2, ISSN:2399-3642

In many RNA molecules, pseudouridines occur at conserved positions in functional sites. A great diversity of pseudouridine synthases guarantees the insertion of the modified base at precise locations. The accepted structural role of pseudouridines is a reduction of the RNA flexibility around the modification site. However, experiments rarely yield clear-cut evidence. The article “Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites” published in 2019 in Biochemical and Biophysical Research Communication by Kennedy et al. constitute a provocative case [1]. This example illustrates how a definite conformational state can be selected through small energy differences in a constrained environment.

RNA modifications are involved in numerous biological processes. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base-pairing formation, protein recognition, RNA structure and stability. tRNAs are the most abundantly modified RNA molecules. Analysis of the roles of their modifications in response to stress, environmental changes, and infections caused by pathogens, has fueled new research areas. Nevertheless, the detection of modified nucleotides in RNAs is still a challenging task. We present here a reliable method to identify and localize tRNA modifications, which was applied to the human pathogenic bacteria, Staphyloccocus aureus. The method is based on a separation of tRNA species on a two-dimensional polyacrylamide gel electrophoresis followed by nano liquid chromatography-mass spectrometry. We provided a list of modifications mapped on 25 out of the 40 tRNA species (one isoacceptor for each amino acid). This method can be easily used to monitor the dynamics of tRNA modifications in S. aureus in response to stress adaptation and during infection of the host, a relatively unexplored field.

International audience

The 1555 A to G substitution in mitochondrial 12S A‐site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G‐mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read‐through of mtDNA‐encoded MT‐CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock‐in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise‐induced hearing damage and anxiety‐related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress‐related behavioral and physiological adaptations.